Supports for antigen/antibody assays and biochemical reactions are typically latex beads, paper, fixed red blood cells and insoluble polymers such as dextran and polystyrene. In these assays and biochemical reactions at least one member of a specific binding pair (hereinafter a "specific binding species") is immobilized on a support and thereafter subjected to chemical reactions, physical manipulation or both in a manner that ultimately results in its binding (transitory or permanent) of the other member of the specific binding pair to the specific binding species. This coupling of the two members of the specific binding pair is useful to detect qualitatively or quantitatively the presence of one of the species in a sample as in a competitive assay, an immunoassay, a protein binding assay. Similarly, the binding of the two members of the specific binding pair can be used to take advantage of the inherent properties of one of the species, e.g., an enzymatic property.
A persistent problem in designing assays and reaction. Systems is the difficulty in immobilizing the specific binding species on the support so that it will withstand washings and remain on the support under the contemplated chemical conditions. Another problem is denaturing the specific binding species pair, i.e., reversing prematurely the binding of the two species such that the bound member disassociates from the immobilized member or the immobilized member and the bound member disassociate from the support. Reactivity of the support under the contemplated conditions of use, resulting in nonspecific binding of constituents in a test sample, is another persistent problem.
Microcapsules have been suggested for use as supports in immune response assays. UK patent Application 2,079,937 A (published 27 Jan. 1982) and UK patent Application 2,079,936 A (published 27 Jan. 1982) both describe making microcapsules with crosslinked wall materials encapsulating an oily core substance. Functional groups with sites for binding an antigen or antibody are attached to the wall by a crosslinking agent.
Fluorocarbon emulsions have been suggested for use as in vivo erythrocyte substitutes. Some fluorocarbon emulsions seem to have good oxygen transport characteristics and appear to be nontoxic and safely metabolized. Other fluorochemical emulsions have been shown to be toxic in laboratory animals or not metabolized and eliminated. Geyer describes various emulsions in Chapter 1, "The Design of Artificial Blood Substitutes", Drug Design, Vol. VII Academic Press, N.Y. (1976) and in "Whole Animal Perfusion with Fluorocarbon Dispensors" Federation Proceedings, Vol. 29 No. 5, p. 1758, (1970). Serum albumin, phospholipids (including lecithin), and surfactants such as Pluronic-F68 (Wyandotte Chemical Corp., Wyandotte, Mich.) have been used as emulsifying agents.
For use as artificial blood Sloviter in U.S. Pat. No. 4,423,007, has suggested emulsions of perfluoro compounds coated with a non-antigenic lipid, preferably egg yolk phospholipid or lecithin, in aqueous medium. He reports In U.S. Pat. No. 4,397,870 that the duration of effective droplet levels in the bloodstream is brief owing to the apparent removal of the lecithin coating and exposure of the perfluoro droplet surface in the bloodstream. Infusion of the patient who has previously received an infusion of an emulsion of coated droplets with the same substance used to coat the perfluoro compound droplet is recommended.
Sloviter in "Erythrocyte Substitute for Perfusion of Brain" Nature Vol. 216, Nov. 1967, 458, has also suggested dispersing a perfluoro compound in a simulated blood plasma compound of 8% bovine serum albumin in Krebs Ringer bicarbonate buffer. After the emulsion was formed and sedimented all soluble protein was washed away. The sedimented material was analyzed and found to contain about 5% protein.
The Japanese have also been active in the field of artificial blood. U.S. Pat. No. 4,252,827 is directed to fluorocarbon compound emulsions that are sufficiently stable to be kept for a long period of time without change in droplet size and can be mixed with plasma extenders such as dextran and hydroxyethyl starch. It describes an emulsion in an organic medium having a perfluorocarbon compound with 9 to 11 carbon atoms, a perfluoro-tert-amine having 9 to 11 carbon atoms, a surfactant having a molecular weight of about 2,000 to 20,000, a phospholipid and at least one fatty acid.
The effect of perfluoro organic compound emulsions on serum proteins and phosolipids has been studied by V. V. Obraztsuv et al. "Binding of Proteins and Phospholipid by Emulsion of Perfluoro Organic Compounds" Ftoruglerodrye Gazoperrnosyaschchie Sridy [531 FA5] 1984 147-52 (Russ). The protocol followed by these authors is not clear. They appear to have analyzed the amount of protein and phospholipids removed from solution only at equilibrium conditions. They do not report any experiments attempting to wash adsorbed protein and phospholipid off the emulsion droplets. They state "irreversible and denaturant character of binding of proteins by the hydrophobic surface raises the question to what extent the concentration of the proteins in the blood might be decreased as a result of the extensive blood substitution with PF0C emulsion."